Dengue (DEN) has become the most significant source of arthropod-borne viral disease in humans. Approximately 2.5 billion people (40% of the world's population) live at risk for dengue exposure across the globe, resulting in more than 100 million cases of dengue related illnesses each year. As a result of the increasing frequency of dengue related illnesses, and the introduction of Aedes mosquitos into parts of the United States, DEN is now classified as a Category A pathogen of interest by the National Institutes of Health. Several lines of evidence support a significant role for antibodies during protection from DEN infection and the establishment of immunity following vaccination. However, the presence of DEN antibodies has also been linked to a more severe clinical outcome due to the ability of antibodies to facilitate DEN infection under some circumstances. This antibody dependent enhancement (ADE) of infection poses a significant challenge for the development of a DEN vaccine and highlights the importance of understanding not only the magnitude, but also the breadth, specificity, and persistence of humoral immunity in response to vaccination. The standard method for detecting neutralizing antibodies to dengue viruses is the plaque reduction neutralization test (PRNT). While use of this methodology has allowed for great insight into flavivirus biology and pathogenesis, it has several major limitations. In this proposal, we will develop a novel approach to detecting and measuring antibodies with neutralizing activity present in human sera. Specific Aim 1: Construction and evaluation of a DEN replicon. Specific Aim 2: Production and characterization of DEN reporter virus particles. Specific Aim 3: Evaluation of the utility of DEN reporter virus particles for the detection of neutralizing antibodies.